High Target Homology Does Not Guarantee Inhibition: Aminothiazoles Emerge as Inhibitors of Plasmodium falciparum

In this study, we identified three novel compound classes with potent activity against Plasmodium falciparum, the most dangerous human malarial parasite. Resistance of this pathogen to known drugs is increasing, and compounds with different modes of action are urgently needed. One promising drug target is the enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) of the methylerythritol 4-phosphate (MEP) pathway for which we have previously identified three active compound classes against Mycobacterium tuberculosis. The close structural similarities of the active sites of the DXPS enzymes of P. falciparum and M. tuberculosis prompted investigation of their antiparasitic action, all classes display good cell-based activity. Through structure–activity relationship studies, we increased their antimalarial potency and two classes also show good metabolic stability and low toxicity against human liver cells. The most active compound 1 inhibits the growth of blood-stage P. falciparum with an IC50 of 600 nM. The results from three different methods for target validation of compound 1 suggest no engagement of DXPS. All inhibitor classes are active against chloroquine-resistant strains, confirming a new mode of action that has to be further investigated.


Docking analysis
For the oxime series, we found that compound 2 was predicted to bind in the ThDP binding site.H-bonding interactions between one of the phenol OH groups and the backbone carbonyl of Val660 as well as between the oxime oxygen atom and the sidechain of His941 were identified (Figure S3).In the predicted binding mode of compound 13, the two aromatic rings have switched positions compared to the binding mode of compound 2 and three H-bonds are now formed with His941, Gly503 and Ser532 (Figure S4).This difference contributes to the higher predicted affinity of compound 2, which again follows the trend observed in the experimental data.For the indole series, we found that compound 3 was predicted to bind in the ThDP binding site.The OH group of the hydroxymethylene moiety was predicted to make a H-bond interaction with Val660 (Figure S5).Compound 33 was predicted to have the same binding mode as compound 3, with the additional fluorine atom contributing positively to the predicted binding affinity (Figure S6), as observed in the experimental data.Inhibition data for all compounds  Our search for compounds 26 and 28 yielded the similar compounds CHEMBL1945694 and CHEMBL570782 (Figure S15), which are reported to inhibit the Enoyl-[acyl-carrier-protein] reductase (ENR or FabI) from Francisella tularensis. 22he two compounds we found belong to a larger class of benzimidazoles, first identified to inhibit F. tularensis FabI in 2012 using a LBVS approach by Johnson and coworkers. (23)In follow-up studies, they reported derivatives with improved activity against the Fabl enzyme of up to 14 nM and determined the crystal structure of FabI from F. tularensis in complex with CHEMBL1945694. (24,25)e FabI enzyme is part of the fatty acid biosynthesis pathway-II (FAS-II) found in a variety of microorganisms, including P. falciparum.(29) The FabI enzyme catalyzes the final reduction step, suppling fatty acids for cell-wall biosynthesis.(36) Therefore, inhibition of FabI cannot explain the activity we observed in the blood stage assays.However, it could be beneficial for a new anti-malarial drug to inhibit FabI as a second target, as FabI is essential for liver stage proliferation and therefore transmission. (36)ken together, the structural similarity of the compounds hints to FabI being an additional target of our hit classes.In particular, the indole-class is very likely to bind to FabI, as there are co-crystal structures of related compounds.Inhibition of Fabl does not explain the observed effects in culture in our assays, as this pathway is only important during the liver stage of P. falciparum growth. (28)man off-target enzymes  *Compounds were checked for PAINS motives using the PAINS filter of the software StarDrop, which searches for functional groups defined in the publication by J.Baell and G. Holloway. (17) Scheme S1.Synthesis of oxime 5 not following general procedures O-B.i) Pyridine hydrochloride, microwave, 15 W, 110 °C, 5 min.

Figure S1 .
Figure S1.Superposition of MtDXPS crystal structure (grey, PDB: 6a9h) with the PfDXPS homology model based on drDXPS structure 2o1x.The core of the enzyme shows a high similarity with the MtDXPS structure.PfDXPS is colored by Cα-RMSD to the MtDXPS structure, from blue = low RMSD via green to red = high RMSD; yellow: no corresponding amino acids in MtDXPS structure.Typical for Plasmodium

Figure S2 .
Figure S2.Superposition of the active site of MtDXPS and PfDXPS.The PfDXPS amino acids are colored by Cα-RMSD to the MtDXPS structure, from blue = low RMSD via green to red = high RMSD; the carbon atoms of the ThDP ligand are shown in gold; heteroatoms are colored in red for oxygen, blue for nitrogen and yellow for sulfur atoms; the green sphere represents a Mg 2+ -ion.Most amino acids are found in identical arrangements, except His941 (corresponding to His416 of MtDXPS) at the bottom right, which is predicted to be in a different rotameric state.

Figure S3 :Figure S4 :
Figure S3: Binding mode and pose diagram of compound 2. Protein surface representation is clipped for clarity.Green and red spheres represent positive or negative contribution to the predicted affinity, respectively.

Figure S5 :Figure S6 :
Figure S5: Binding mode and pose diagram of compound 3. Protein surface representation is clipped for clarity.Green and red spheres represent positive or negative contribution to the predicted affinity, respectively.

Figure S13 .
Figure S13.Target verification using PfTPK overexpressing parasites (3D7-DXPS + ).Antiplasmodial activity of compounds (A: compound 1, B: compound 2, C: compound 3) against PfTPK overexpressing parasites (blue squares) in comparison to the MOCK cell line (black triangles).Data represent the result from one experiment with technical triplicates (error bars indicate SEM).For IC 50 determination, data was analyzed using nonlinear regression of the log-concentration-response curves and interpolated from the sigmoidal curve.

Table S1 .
Solubility in PBS buffer and 2% DMSO of new oxime derivatives in comparison to the original hit 2.

Table S2 :
Estimated affinity values for docked compounds.

Table S3 .
IDP rescue assay: IC 50 values with and without IDP against P. falciparum 3D7 and NF54.SD is displayed as error measure when more than one experiment was performed.a = mean of triplicates, b = mean of duplicates.

Table S5 . Compound evaluation against P. falciparum 3D7-TPK + and P. falciparum 3D7-DXPS + in comparison to MOCK cell line.
The table displays the IC 50 values from antiplasmodial screens using P. falciparum 3D7-PfTPK + (3D7-TPK + ) and Plasmodium falciparum 3D7-PfDXPS (3D7-DXPS + ) overexpression cell lines, as interpolated from nonlinear regression curves.95% CI is displayed as error measure.Percentages indicate statistical probability of the simpler model "LogIC 50 same for all data sets" being correct.

Table S6 .
Summary of determined IC 50 values of all compounds.The numbers I-III are indicating the used method.

Table S7 .
Summary of possible human off-target enzymes encountered during the search for bacterial targets.The molecule drawn in "search hit" is reported in the cited source to be targeting the enzyme or pathway given in "target enzyme".Only one hit molecule is shown, the cited source often reports many more derivatives.No ranking and in-depth analysis of likelihood to be a target was performed.